All P2X receptors transduce significant Ca2+ currents at the resting membrane potential that trigger many of the physiological and pathophysiological actions of extracellular ATP. The molecular physiology of this Ca2+ current is poorly understood despite significant recent advances in functional and structural studies. In this proposal, we focus on the molecular physiology of the Ca2+ current of native and recombinant P2X7 receptors (P2X7Rs). P2X7Rs are prominently expressed in hematopoietic cells and play an essential role in inflammation. They are also found in bone, neurons, and glia where they influence differentiation, homeostasis, and degeneration. Regrettably, meaningful gaps remain in the P2X7R literature despite 40+ years of intensive study. In the experiments outlined in the three specific aims of this proposal, we probe two unknowns. First, we seek to identify the molecular determinates of the greater-than-expected Ca2+ flux of most P2X7Rs. In Specific Aim 1, we use patch-clamp photometry to identify specific domains responsible for the Ca2+ flux. This aim is significant because the structural models derived from crystals of truncated P2XRs may present a distorted view of the permeation pathway and fail to define the ion selectivity filter. In Specific Aim 2, we investigate the curious finding that naturally occurring splice variants of P2X7Rs with distinct N-termini but identical pore-forming helices transduce dramatically different Ca2+ currents, suggesting that the N-termini, which move during gating, play a significant role in positioning an intra-pore Ca2+ selectivity filter. These data leave open the possibility that drugs and signaling complexes that interact with the N-terminus might selectively modulate the physiologically important Ca2+ flux through the channel. Second, we seek to understand the biophysical basis of the time-dependent changes in ATP-current reversal seen during long applications of agonist. Traditionally thought to reflect a gradual dilation of the pore, a recent report suggests that the reversal actually occurs as ions redistribute across the cell surface membrane. In Specific Aim 3, we use novel wild- type and mutant receptors to test the hypothesis that pore dilation and ion accumulation are not mutually exclusive phenomena. These experiments are important because genuine pore dilation could impact Ca2+ homeostasis in living cells. That is, the modified channel structure responsible for dilation could disrupt a key Ca2+ binding site within the P2X7R pore, leading to a reduction in Ca2+ influx and a change in extracellular ATP- dependent cell physiology.